The central objective of this application is to understand how self reactive T cells develop in non obese diabetic (NOD) mice during disease progression. It has been suggested that insulitis and subsequent diabetes is induced by an immunoregulatory imbalance of autoreactive T cells, such as a dominance of Th1 over Th2 T-cell subset function and cytokine production. However, what has been lacking in the current work is the ability to follow an antigen specific T cell response in vivo as the disease progresses. In order to test this hypothesis, and determine the mechanism underlying this loss of tolerance, we propose to use peptide/MHC multimers which have been developed to stain T cells in antigen specific manner to follow T cell responses to two beta cell antigens-GAD (glutamate decarboxylase) and insulin in vivo as the disease progresses. In spite of the perceptible differences in the development of GAD and insulin specific T cells. both have been proposed to mark a key turning point in the development of IDDM in NOD mice. In addition, tolerization with either GAD or insulin in pre diabetic mice greatly reduces the incidence of disease. Thus, by tracking these T cells before and during disease progression, and following therapeutic intervention, we hope to achieve the following goals: (i) document when these auto-reactive T cells develop and where they reside; (ii) determine whether or not there is a change in their cytokine responses to antigens, and if so, what are the changes and when do they occur; and (iii) what are the characteristics of these T cells when NOD mice are tolerized with these proteins. A clear understanding of the development of self-reactive T cells in NOD mice will provide important new insights into how a T cell mediated auto- immunity develops. Such information can then be used to develop and test protocols for therapeutic interventions.